The ultimate success of immunotherapy for brain malignancies, such as malignant glioma, will require integration of in-depth understanding of immunology with solutions for the following long-standing challenges: 1) paucity and heterogeneous expression of glioma-specific antigens; 2) poor homing and persistence of effector cytotoxic T lymphocytes (CTLs); and 3) glioma-induced immunosuppression. My laboratory has been contributing to critical discoveries in each of these areas, and integrated our findings into novel immunotherapy clinical trials for glioma patients. In the current proposal, we will leverage our current research directions by combining our expertise on glioma antigens and cutting-edge cell-engineering technologies in preclinical studies. We hypothesize that integration of novel cell-engineering and antigen-targeting approaches will allow us to develop safer and more effective immunotherapy strategies by overcoming heterogeneous expression of antigens and unique challenges in brain immunology. We will evaluate the following strategies: 1. Novel glioma neoantigens for safe and effective immunotherapy. We will leverage our current NINDS awards (R01NS096954 and R21NS093654) and characterize T-cell receptors (TCRs) specific to neoantigens derived from both pediatric and adult gliomas. 2. Sequential chimeric antigen receptor (CAR)/TCR system for targeting multiple antigens. As a way to safely target glioma-associated antigens (GAAs) in the tumor microenvironment without damaging normal cells outside of the brain, we will evaluate the novel sequential Synthetic Notch (synNotch) CAR/TCR system, in which antigen signaling through the first CAR or TCR against a tumor-specific antigen induces the second, anti-GAA CAR/TCR to trigger the CTL activity at the tumor site. 3. Targeting the glioma immune environment by creating tertiary lymphoid organs (TLOs). The absence of lymphatic organs and professional antigen presenting cells are thought to be major reasons for insufficient immune responses in the brain. We will evaluate whether induction of TLOs in the brain tumor site will facilitate efficient and long-lasting glioma antigen-specific immune responses in the brain tumor site. These 3 strategies will be logically integrated into combination approaches. Novel antigens and TCRs will be adopted into the synNotch CAR/TCR system, and the TLO approach would also be most beneficial when combined with the synNotch CAR/TCR system. As expected per the purpose of the NINDS R35 mechanism, these strategies may involve high risks. However, based on our proof-of-principle preliminary data, we will persistently pursue our goals with the long-term support by the R35 mechanism, and flexibly and swiftly adopt new technologies. These studies will also integrate with other areas of ongoing studies in our lab. For example, oncolytic virus-mediated expression of target antigen and CTL-attracting chemokine (?payload? approaches) would help us to overcome the paucity and heterogeneous expression of antigens as well as tumor homing of CTLs to the glioma tissue. The R35 would allow these integrations.